Process for the production of beta-carotene



United States Patent 14 Claims. (:1. 19s 2s The present inventionrelates to the production of ,B-carotene by fermentation.

fl-Carotene can be obtained by the submerged fermentation ofmicroorganisms of the Choauephora or Blakeslea type. A variety ofconditions favour the production of ,B-carotene. Barnett et val.[Science, 123, 141 (1956)] showed that the production of fi-carotene isimproved by the simultaneous culture of opposite and forms of onespecies. The culture of opposite forms of different species also leadsto an improved yield [C. Hesseltine, Mycologia, 49, 449 (1957)]. It hasalso been found that the addition to the nutrient medium of whole orhydrolysed grain, vegetable oils, surface-active agents, antioxidants orthickening agents increases the yield of B- carotene [R. Anderson etal., J. Agr. Food Chem. 6, 543 (1958); A. Ciegler et al., App. Microb.7, 94 and 98 (1959)]. Moreover, Mackinney et a1. [J. Amer. Chem. Soc.74, 3456 (1952)] have shown that the addition of fi-ionone to the staticculture of a Phycomyces greatly increases the formation of B-carotene,to the detriment of the formation of other carotenoid pigments. Andersonet al. (loc. cit.) noted the same effect in agitated cultures ofBlakeslea and Choanephora.

It has now been found that it is advantageous to add2,6,6-trimethyl-1-acetylcyclohexene to the nutrient medium. Thiscompound acts as a precursor for S-carotene, but has the advantage overS-ionone, heretofore used for the same purpose, of being less complexand therefore more readily obtainable. According, therefore, to thepresent invention, a process for the production of fi-carotene comprisesculturing aerobically the and forms of Blakeslea zrispora (NRRL 2456 and2457) in a nutrient medium containing 2,6,6 trimethyll-acetylcyclohexene.

The addition of 2,6,6-trimethyl-l-acetylcyclohexene to the nutrientmedium leads to a considerable increase in the ,B-carotene yield, andmay be made instead of, or in addition to, an addition of j3-ionone.When 2,6,6-trimethyl-l-acetylcyclohexene is used alone, it may be addedto the culture medium in an amount from 0.1 to 10 g. per litre, at thebeginning of or during the course of the fermentation, in one or morelots. When 2,6,6-trimethyll-acetylcyclohexene is employed in associationwith 3- iouone, the quantity of the former is then ordinarily 0.1 to 6g. per litre, and of the latter 0.1 to 4 g. per litre. Preferably, thereis employed either between 0.5 and 2 g. per litre of2,6,6-trimethyl-l-acetylcyclohexene alone, or a mixture such that themedium contains 0.3 to 1.5 g. per litre of2,6,6-trimethyl-1-acetylcyclohexene and 0.2 to 1 g. per litre offi-ionone, the addition being made 1 to 3 days after the beginning ofthe culture. Regardless, however, of the quantity of these compoundsadded, and of the moment of the addition, it is desirable to continuethe culture for 6 to 10 days after the inoculation in order to obtainthe maximum yield of B-carotene.

The culture medium may vary, but essentially it contains assimilablesources of carbon and nitrogen, mineral elements and, optionally, growthfactors, anti-oxidants, surface-active agents, thickening agents andother precursors.

As assimilable carbon source, there may be used carbohydrates such asglucose, dextrins, starch, or animal or vegetable oils such as lard orsoya bean oil or cottonseed oil. The assimilable nitrogen source may bepure chemical substances or complex substances containing mainlynitrogen in protein form, e.g. casein, lactalbumin, and gluten and theirhydrolysates, soya bean flour, peanut flour, yeast extracts, distillerssolubles and cornsteep liquor.

Some of the mineral elements added, such as the alkali metal, oralkaline-earth metal phosphates, may have a buffering or neutralisingeffect.

The most frequently employed and most valuable of the growth factors isvitamin B or thiamine. As antioxi dants, there may especially bementioned N,N'-di-phenyl paraphenylenediamine, more2,2,4-trimethyl-6-ethoxy-1,2- di-hydroquinoline, ascorbic acid andsorbic acid, the two first being preferred. The surface-active agentsare preferably of the non-ionic type, such as derivatices of sorbitolwith fatty acids, or products based upon ethylene oxide condensates.Among the most commonly employed thickening agents are starch,carboxymethylcellulose and agar.

It is also preferred that the nutrient medium contains one or more ofthe activators disclosed in our application Serial No. 285,574, filed onJune 5, 1963, now Patent No. 3,235,467. Such an activator is eitherdimethylformami-de, acetamide, dimethylacetamide, propionamide,butyramide, succinimide, N-bromosuccinimide, N-hydroxymethylsuccinimide,caprolactam, a-pyrrolidone, or dimethylsulphoxide. The preferredactivators are a-pyrrolidone and succinimide.

The following examples show how the invention may be put into practice.

Example) I Culture medium A is prepared as follows. 500 cc. of

water containing 60 g. of distillers solubles are boiled for 15 minutes.After cooling, there are added:

The volume is made up to 1000 cc. with distilled water. The mixture isadjusted to a pH value of 6.3 with a few drops of 10 N sodium hydroxide,distributed in 300-cc. Erlenmeyer flasks in a proportion of 50 cc. perflask, and then sterilised for 20 minutes at C. After sterilisation andcooling 0.5 cc. of a sterile 1% solution of 2,2,4-trimethyl-6-ethoxy-1,Z-dihydroquinoline in petroleum is added to eachflask under sterile conditions.

Each flask is then inoculated with 5 cc. of an agitated culturecontaining the and forms of Bla-kcslea trispora (NRRL 2456 and NRRL2457) and aged for 48 hours. The flasks :are then placed on a rotativeshaker table turning at 220 r.p.m. in a thermostat at 26 C. Afterincubation for 2 days, the flasks are divided into two groups and theyreceive the following additions under sterile conditions:

1st group: petroleum 0.5 cc. 2nd group: 50 mg. of 2,6,6-trimethyl-1acetylcyclohexene (TMACH) in solution in 0.5 cc. of petroleum.

The cultures are then continued under the same temperature and stirringconditions for 8 more days. At the end of this time, the production ofB-carotene is maximum in all the flasks.

The determination of the B-carotene is carried out as follows: Themycelium is separated by filtration, washed with water and then driedfor one night at 35 C. in vacuo. The dry mycelium is then extracted withhexane. The ,B-carotene is separated from the other carotenoids presentby chromatography of the extract on alumina. The elution fractionscontaining ii-carotene are combined and spectre-photometricallydetermined in relation to a pure specimen of fl-carotene. The followingresults are obtained: Mg./l. of B-carotene 1st group, without precursor825 2nd group, with TMACH 1265 Example II A culture medium B is preparedin the same manner as medium A in Example I, but 200 mg. of2-pyrrolidone are added under sterile-conditions to each flask, after,sterilisation and cooling, in addition to the antioxidant in solution inpetroluem. The flasks are then inoculated and incubated under theconditions described in Example I. After incubation for 2 days, theflasks are divided into two groups and they receive the followingadditions under sterile conditions:

1st group: petroleum, 0.5 cc. 2nd group: 50 mg. of2,6,6-trimethyl-l-acetlycyclohexene (TMACH) in solution in 0.5 cc. ofpetroleum.

After these additions, the cultures are continued for 8 more days underthe same temperature and stirring conditions.

The B-Carotene content is determined as in Example I, and the followingresults are obtained:

Mg./l. of fi-carotene 1st group, without precursor 1360 2nd group, withTMACH 1820 Example III A culture medium C is prepared in the same manneras medium A in Example I, adding 4 g./l. of succinimide as well as thestarch, oils and other constituents. After distribution in Erlenmeyerflasks and sterilisation, the medium is similarly completed by a sterileaddition to each flask of antioxidant in solution in petroleum. Theflasks are thereafter inoculated and incubated under the conditionsdescribed in Example I. After incubation for 2 days, the flasks aredivided into two groups and they receive the following additions understerile conditions:

1st group: petroleum, 0.5 cc. 2nd group: 50 mg. of2,6,6-trimethyl-1-acetylcyclohexene (TMACH) in solution in 0.5 cc. ofpetroleum.

After these additions, the cultures are continued for 8 days under thesame temperature and stirring conditions.

The fi-cairotene contents are determined as in Example I, and thefollowing results are obtained:

Mg./l. of fl-carotene 1st group, without precursor 1240 2nd group, withTMACH 1690 Example IV Medium C, including the antioxidant in petroleum,is prepared as in Example III and the flasks containing the medium areinoculated and incubated under the conditions described in Example I.After incubation for 2 days, the flasks are divided into seven groupsand each flask receives the following additions under sterileconditions:

1st group: 50 mg. of TMACH in solution in 0.5 cc. of

petroleum. 2nd group: 50 mg. of TMACH and 10 mg. of B-ionone in solutionin 0.5 cc. of petroleum.

3rd group: 50 mg. of TMACH and 20 mg. of B-ionone in solution in 0.5 cc.of petroleum.

4th group: 50 mg. of TMACH and 30 mg. of fi-ionone in solution in 0.5cc. petroleum.

4 5th group: 50 mg. of TMACH and 40 mg. of ,B-ionone in solution in 0.5cc. of petroleum, 6th group: 50 mg. of TMACH and 50 mg. of p-ionone insolution in 0.5 cc. of petroleum. 7th group: 50 mg. of )S-ionone insolution in 0.5 cc. of

petroluem.

After these additions, the cultures are continued for 8 more days underthe same temperature and stirring conditions.

The fl-carotene contents are determined as indicated in Example I, andthe following productions are obtained:

58 cc. of 10 N sodium hydroxide and the medium is completed with thefollowing materials:

Starch g 750 Soya bean oil cc 450 Cottonseed oil cc 450 Yeast extract g15 Monopotassium phosphate g 7.5 Manganese sulphate, monohydrate g 1.52,2,4-trimethyl-6-ethoxy-1,2-dihydroquinoline in 50% solution in ethanolcc 3 Aqueous thiamine hydrochloride solution "(0.3

The volume is adjusted to 15 litres with water. The pH of the mixture is6.45. The medium is sterilised for 50 minutes at 122 C. by steaminjection. After cooling, the pH is 5.95 and the volume 14.5 litres. Asolution of 45 g. of succinimide in 350 cc. of water, and then cc. ofpetroleum filtered under sterile conditions are added under sterileconditions. The fermentation vessel is then inoculated with 1500 cc. ofan inoculum culture of the and forms of Blakeslea trispora, aged for 49hours. The culture is carried out at 26 C. with agitation with a turbinerotating at 400 r.p.m. and with aeration with sterile air supplied at1.2 cubic metres per hour.

After 40 hours of culture, 150 cc. of petroleum are added under sterileconditions, and a device is set in operation which adds continuously tothe culture, between the 2nd and 6th days of the fermentation, 1000 cc.of sterile aqueous solution containing 450 g. of glucose monohydrate.Thesame aeration, agitation and temperature conditions are maintainedfor the 5 days during which'the culture is continued.

In two other 30-1itre fermentation vessels, the abovedescribed medium isprepared in the same way and completed with the same additions. Themedium is inoculated with the same inoculum culture and the culture iscarried out throughout the duration of the fermentation (7 days) underthe above-described aeration, agitation and temperature conditions.After 40 hours of culture, there are set in operation in the twoapparatus the devices for the continuous addition of glucose monohydrateunder the' conditions to the second fermentation vessel: 15 g. of 2,6,6-trimet-hyl-l-acetylcyclohexene (TMACH) in sterile solution in 150 cc.of petroleum (i.e. 1 g./l.); and to the third fermentation vessel: 22.5g. of 2,6,6-trimethyl-1-acetylcyclohexene in sterile solution in 150 cc.of petroleum (i.e. 1.5 g./l.).

The determination of B-carotene produced in these three fermentationvessels is carried out as in Example I. The following results areobtained:

Mg./l. of fi-carotene Operation without percursor 770 Operation withaddition of TMACH (1 g./l.) 1280 Operation with addition of TMACH (1.5g./l.) 1445 We claim:

1. Process for the production of fl-carotene which comprises culturingaerobically the and forms of Blakeslea trispora, NRRL 2456 and 2457, ina nutrient medium containing 0.1 to g. per litre of2,6,6-trimethyl-l-acetylcyclohexene and recovering fi carotene from thesaid medium.

2. Process for the production of fi-carotene which comprises culturingaerobically the and forms of Blakeslea trispora, NRRL 2456 and 2457, ina nutrient medium containing 0.1 to 6 g. per litre of2,6,6-trimethyl-l-acetylcyclohexene and 0.1 to 4 g. per litre offi-ionone and recovering ,B-carotene from the said medium.

3. Process according to claim 1 in which the nutrient medium contains0.5 to 2.0 g. per litre of 2,6,6-trimethyll-acetylcyclohexene.

4. Process according to claim 2 in which the nutrient medium contains0.3 to 1.5 g. per litre of 2,6,6-trimethyll-acetylcyclohexene and 0.2 to1 g. per litre of fl-ionone.

5. Process according to claim 3 in which the addition of the2,6,6-trimethyl-l-acetylcyclohexene is made 1 to 3 days after thebeginning of the culture.

6. Process according to claim 4 in which the addition of the2,6,6-trimethyl-l-acetylcyclohexene and fi-ionone is made 1 to 3 daysafter the beginning of the culture.

7. Process according to claim 1 in which the nutrient medium alsocontains thiamine.

8. Process according to claim 2 in which the nutrient medium alsocontains thiamine.

9. Process according to claim 1 in which the nutrient medium alsocontains an antioxidant selected from the group consisting of2,2,4-trimetl1yl-6-ethoxy-1,Z-dihydroquinoline andN,N-diphenyl-p-phenylene diamine.

10. Process according to claim 2 in which the nutrient medium alsocontains an antioxidant selected from the group consisting of2,2,4-trimethyl-6-ethoxy-1,2-dihydroquinoli-ne andN,N'-diphenyl-p-phenylene diamine.

11. Process according to claim 1 in which the nutrient medium containsan activator selected from the group consisting of dimethylformamide,acetamide, dimethylacetamide, propionamide, butyramide, succinimide,N-bromosuccinimide, N-hydroxymethylsuccinimide, caprolactam,u-pyrrolidone, and dimethylsulphoxide, the said activator being presentin said nutrient medium in amount from 0.1 to 10 g. per litre.

12. Process according to claim 1 in which the nutrient medium containsan activator selected from the group consisting of u-pyrrolidone andsuccinimide in amount from 0.5 to 6 g. per litre of said nutrientmedium.

13. Process for the production of ,B-carotene which comprises culturingaerobically the and forms of Blakeslea trispora, NRRL 2456 and 2457, ina nutrient medium containing assimilable sources of carbon and nitrogen,thiamine, an antioxidant selected from the group consisting of2,2,4-trimethyl-6-ethoxy-1,2-dihydroquinoline andN,N'-diphenyl-p-phenylene diamine, an activator selected from the groupconsisting of u-pyrrolidone and succinimide in amount from 0.5 to 6.0 g.per litre of nutrient medium, and 0.5 to 2.0 g. per litre of nutrientmedium of 2,6,6-trimethyl-l-acetylcyclohexene, the last named compoundbeing added 1 to 3 days after the beginning of the culture, and theculture being continued for a total of 6 to 10 days and recoveringB-carotene from the said medium when the culture is complete.

14. Process for the production of fi-carotene which comprises culturingaerobically the and forms of Blakeslea trispora, NRRL 2456 and 2457, ina nutrient medium containing assimilable sources of carbon and nitrogen,thiamine, an antioxidant selected from the group consisting of2,2,4-trimethyl-6-ethoxy-1,2-dihydroquinoline andN,N-diphe-nyl-p-phenylene diamine, an activator selected from the groupconsisting of a-pyrrolidone and succinimide in amount from 0.5 to 6.0 g.per litre of nutrient medium and 0.3 to 1.5 g. per litre of nutrientmedium of 2,6,6-trimethyl-l-acetyl-cyclohexene and 0.2 to 1.0 g. perlitre of said medium of B-ionone, the two last named compounds beingadded 1 to 3 days after the beginning of the culture, and the culturebeing continued for a total of 6 to 10 days and recovering fi-carotenefrom the said medium when the culture is complete.

No references cited.

A. LOUIS MONACELL, Primary Examiner.

ALVIN E. TANENHOLTZ, Examiner.

1. PROCESS FOR THE PRODUCTION OF B-CAROTENT WHICH COMPRISES CULTURINGAEROBICALLY THE + AND - FORMS OF BLAKESLEA TRISPORA, NRRL 2456 AND 2457,IN A NUTRIENT MEDIUM CONTAINING 0.1 TO 10 G. PER LITRE OF2,6,6-TRIMETHYL-1-ACETYLCLCLOHEXENE AND RECOVERY B-CAROTENE FROM THESAID MEDIUM.